Three-dimensional single-cell imaging analysis of tumor-natural killer cell interaction dynamics in multi-cell-type tumor organoid models

Background
Patient-derived tumor organoids have emerged as physiologically relevant models that better recapitulate primary tumor architecture and function compared with traditional cancer cell lines and mouse tumor models. Despite their advantages, standardized protocols for establishing complex organoid systems that incorporate both tumor and key non-tumor components of the tumor microenvironment (TME) remain limited. Most of the available literature focuses on organoid-T cell co-cultures.

Methods
Here we present a detailed and reproducible protocol for generating organoid-Natural Killer (NK) cell co-culture models, specifically a three-component model system comprising patient-derived tumor organoids, primary NK cells, and endothelial cells. The protocol also includes a workflow for three-dimensional (3D) live-cell imaging to visualize and quantify real-time interaction dynamics between NK cells and tumor organoids at the single cell level.

Results
Application of this protocol enables quantitative analysis of NK cell-mediated cytotoxicity towards diverse tumor organoid subtypes, both in the presence and absence of endothelial cells. These analyses can reveal distinct NK cell-sensitive, NK cell-resistant, and NK cell-excluded tumor phenotypes within the TME.

Conclusions
This protocol establishes a robust 3D co-culture framework for profiling NK cell responses in complex TMEs. It supports mechanistic studies of immune-tumor interactions and provides a viable preclinical platform for evaluating NK cell-based immunotherapies in a patient-specific context.